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Could Testosterone Replacement Therapy in Hypogonadal Men Ameliorate Anemia, a Cardiovascular Risk Factor? An Observational, 54-Week Cumulative Registry Study
Author's Accepted Manuscript Could Testosterone Replacement Therapy in Hypogonadal Men Ameliorate Anemia, a Cardiovascular Risk Factor? An Observational, 54-week Cumulative Registry Study Li Tao Zhang , Yu Seob Shin , Ji Yong Kim , Jong Kwan Park
PII: DOI: Reference:
S0022-5347(15)05150-2 10.1016/j.juro.2015.10.130 JURO 13046
To appear in: The Journal of Urology Accepted Date: 15 October 2015 Please cite this article as: Zhang LT, Shin YS, Kim JY, Park JK, Could Testosterone Replacement Therapy in Hypogonadal Men Ameliorate Anemia, a Cardiovascular Risk Factor? An Observational, 54week Cumulative Registry Study, The Journal of Urology® (2015), doi: 10.1016/j.juro.2015.10.130. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
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Could Testosterone Replacement Therapy in Hypogonadal Men Ameliorate Anemia, a
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Cardiovascular Risk Factor? An Observational, 54-week Cumulative Registry Study
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Li Tao Zhang,* Yu Seob Shin,* Ji Yong Kim, Jong Kwan Park†
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Department of Urology, Chonbuk National University and Research Institute of Clinical
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Medicine of Chonbuk National University-Biomedical Research Institute and Clinical Trial
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Center of Medical Device of Chonbuk National University, Jeonju, 561-712, Republic of Korea.
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*
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Enclosed: 8 figures and 1 table
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Word count of text/abstract: 2499/250
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Equal study contribution
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Running Title: Effect of testosterone undecanoate therapy on CVD risk factors.
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†
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Jong Kwan Park, M.D., Ph.D.,
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Department of Urology, Chonbuk National University and Research Institute of Clinical
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Medicine of Chonbuk National University-Biomedical Research Institute and Clinical Trial
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Center of Medical Device of Chonbuk National University, Jeonju, 561-712, Republic of Korea.
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Tel.: + 82 (0) 63 250 1510
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Fax: + 82 (0) 63 250 1564
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Correspondence to:
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E-mail: [email protected]
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Purpose: This study was to investigate if testosterone undecanoate in patients with
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hypogonadism attenuates anemia and the risk for cardiovascular disease.
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Materials and Methods: A registry study consisted of 58 participants with subnormal total
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testosterone level (<2.35 ng/ml) and at least mild symptoms of testosterone deficiency. All the
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patients were injected with 1,000 mg of testosterone undecanoate on initial visit, followed by
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injection at 6, 18, 30, 42 and 54 weeks. Serum hormones, Hb, Hct, anemia risk factors, lipid
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profiles, whole blood viscosity and anthropometry were measured.
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Results: Total testosterone (from 1.87 ± 1.09 to 5.52 ± 1.92 ng/ml, p <0.001) and free
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testosterone (from 3.04 ± 2.03 to 7.23 ± 2.90 pg/ml, p <0.001) were restored by testosterone
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undecanoate therapy. Hb and Hct significantly increased after testosterone undecanoate therapy
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by an average of 2.46 g/dl (p <0.001) and 3.03% (p <0.001), respectively. Prevalence of anemia
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(from 29.6 to 10.0%) significantly decreased (p <0.001) and patients with anemia showed a
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significant increase in erythropoietin (p = 0.047) after testosterone undecanoate therapy.
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Reduction in total cholesterol (from 165.89 ± 39.16 to 153.80 ± 154.27 mg/dl, p = 0.002), higher
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whole blood viscosity and increased Hct were observed until 54 weeks compared with baseline,
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however whole blood viscosity and Hct stabilized after 18 weeks.
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Conclusions: 54-week testosterone undecanoate decreased the prevalence of anemia and
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components of metabolic syndrome. A longer duration testosterone undecanoate therapy of more
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than 18 weeks may be effective and safe in reducing the blood viscosity and improving anemia.
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Key Words: anemia; cardiovascular diseases; hypogonadism; whole blood visccosity; testosterone
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MALE hypogonadism results from failure of testes to produce adequate sex hormones; this may
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be because of distant or local hormonal deficiency.1 Normal aging could be one of the causes of
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the deficiency. Collectively referred to as TDS, the associated symptoms may be erectile
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dysfunction, increased abdominal fat and reduced muscle.2 Low circulating testosterone is also
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linked to conditions such as anemia, metabolic syndrome, and CVD.3
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From the Massachusetts Male Aging Study of older men (years 1987-2004), the average
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age-related reduction in TT is 0.8~1.6% per year after age 40.4 For hypogonadal men, anemia is
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a frequent finding as testosterone stimulates erythropoiesis in a dose dependent manner and more
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prominently in older men.5 This is despite the effect being independent of actual erythropoietin
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and transferrin receptor levels.5-7 It is reasonable to hypothesize that a decline in testosterone
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levels in males with TDS may lead to an anemia and increase the risk for CVD.
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The mechanism linking TDS with metabolic sydrome seems to be complicated and multi-
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directional.8,9 It is known that metabolic sydrome is associated with an increasing risk of CVD.
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Hematological parameters such as increased whole blood viscosity (WBV), increased
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erythrocyte aggregation, reduced erythrocyte deformability, and altered erythrocyte morphology
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have also been associated with metabolic syndrome and CVD.10-12
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The aims of the current study were 1) to investigate whether a TU formulation, Nebido®,
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has a sustained benefit on anemia, metabolic and CVD parameters in men with TDS; and 2) to
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measure any dynamic alterations in blood viscosity by TU and its relationship to the risk of CVD
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in patients with TDS. Nebido® was approved as AVEED (3 ml of TU) by the FDA in the United
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State.
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MATERIALS AND METHODS
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Ethics and Informed Consent
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This study was conducted at Chonbuk National University Hospital. Written informed consent
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was obtained from all subjects before study enrollment. Study protocols and informed consent
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forms were approved by the Institutional Review Board (IRB; Number, CUH 2015-06-133-001)
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(Clinical Research Information Service; CRIS, KCT0001680).
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Study Design
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This study was an open-labeled, cumulative analysis of adult males with TDS. The study was
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conducted from February 2012. Last subject completed the study at June 2015. Parameters were
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measured during the initial screening visit (visit 1), followed by measurements at 6, 18, 30, 42,
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and 54 weeks after the initial injection (fig. 1).
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Subjects
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The study population was comprised of hypogonadal patients who were elected to participate in
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the open-label, cumulative registry study. The subjects were male aged 18 to 80 years and had a
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body mass index of ≥ 18 to ≤ 40 kg/m2. The individuals had to have a serum TT level < 2.35
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ng/ml and at least mild TDS to be included in the study. Most hypogonadal men had not received
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any previous androgen treatment, and those who had, had to have had discontinued testosterone
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treatment for at least 6 months prior to enrollment in the study. The patients had a rectal
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examination, transrectal ultrasonography and their prostate-specific antigen levels were checked
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before treatment.
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The enrolled group (n = 58) received active treatment during the open phase of the study. 29
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patients of them were dropped out before visit 3 due to low economic state (22), or without
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improvement of symptoms (7). The patients were injected with 1,000 mg of TU on ventro-
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gluteal muscle of hip on initial visit, followed by injection at 6, 18, 30, 42 and 54 weeks.
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The exclusions were for: tobacco smokers, prostate-specific antigen level of >2.5 ng/ml, a
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current or previous diagnosis of prostate cancer, International Prostate Symptom Score of >15,
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an untreated prolactinoma, Hct of >52% or Hb >18 g/dl, having a uncontrolled diabetes mellitus
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(HbA1c above 10%), having liver and kidney disease, having known diseases of the testis or the
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pituitary.
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General and Biochemical Analyses
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A venous blood sample was drawn for complete blood count, TC, triglyceride, high density
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lipoprotein, LDL, fibrinogen, glucose, hs-CRP, and erythrocyte sedimentation rate (ESR).
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Measurement of Hormones
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TT evaluations were always made between 8 and 10 am. Hormonal tests included TT (COAT-A-
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COUNT, Siemens Healthcare Diagnostics Inc, Los Angeles, CA, USA), FT (Free Testosterone,
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COAT-A-COUNT, Siemens Healthcare Diagnostics Inc,), and estradiol (Estradiol, BECKMAN
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COULTER, Prague, Czech). TT level <2.35 (2.36< TT< 9.96 ng/ml, FT <8.7 (8.8< FT <27)
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pg/ml were considered to be low. The reference range of estradiol is >7.6 and <42.6 pg/ml.
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Measurement of Whole Blood Viscosity
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Viscosity measurement was made at shear rates of 1, 2, 5, 10, 50, 150, 300, and 1,000 sec-1
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samples.13 These shear rates include the velocity of blood flows in the circulatory system.
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Anemic Outcome Measures
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Blood sample levels of iron, ferritin, unsaturated iron binding capacity, total iron binding
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capacity, vitamin B12, folate and erythropoietin were determined.
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Other Main Outcome Measures
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Anthropometry parameters were determined by body composition analyzer.
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Statistical Analysis 7
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Only patients who had completed 54 weeks of treatment were eligible for evaluation.
Statistical analysis was performed using the Statistical Package for Social Sciences for
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Windows, version 21 (SPSS, Chicago IL, USA). All values were checked for normal distribution
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by the Kolmogorov-Smirnov for goodness of fit. Descriptive variables are presented as mean ±
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standard deviation. One way analysis of variance was calculated over time, which was followed
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by the Duncan’s post-hoc test for intergroup comparison if an overall level of significance of p
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<0.05 was reached.
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RESULTS
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Demographics and Comorbidities of the Participants at Baseline
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The 29 subjects completed the 54-week intervention and were assessed at baseline (visit 2), after
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6 weeks (visit 3), 18 (visit 4), 30 (visit 5), 42 (visit 6), 54 (visit 7), and 1 week after
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discontinuation of intervention (visit 8). A number of comorbidities in the patients were reported
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in this registry study. From among the 58 randomized patients there were 27 cases (46.6%) with
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comorbidities. 29.31% of these had type 2 diabetes mellitus, 37.93% had hypertension, 19.0%
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had hypertension and type 2 diabetes together and 22.4% had coronary artery diseases. A small
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percentage (<10%) suffered from elevated triglycerides (4.9%) and dyslipidemia (5.2%). During
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the study, there was no patient who need to receive prostate biopsy for work up of prostate
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cancer. Also, there were no major side effects occurred during the study. Baseline demographic
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and clinical characteristics are shown in table 1.
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Serum Total, Free Testosterone, and Estradiol Profile over 54 Weeks after Treatment
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Serum TT, FT, and estradiol values over the 54 weeks (after visit 7) are shown in figure 2. Pre-
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dose concentrations were 1.87 ± 1.09 ng/ml, 3.04 ± 2.03 pg/ml and 25.22 ± 2.84 pg/ml,
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respectively. After first application of testosterone, TT and FT reads rose rapidly, reaching 5.99
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± 1.97 ng/ml (p <0.001) and 9.13 ± 3.41 pg/ml (p <0.001), respectively. Levels decreased
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linearly from week 6 to a value of 5.45 ± 2.47 ng/ml before the third injection. Thereafter, serum
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TT and FT concentrations slightly altered, but still remained the eugonadal range (2.36-9.96
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ng/ml and 8.8-27 pg/ml) until 54 weeks post-application. Estradiol slowly and significantly
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increased, reaching 25.22 ± 14.77 pg/ml (p <0.001) at visit 4.
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Effects of Testosterone Therapy on Hemoglobin, Hematocrit, and Whole Blood Viscosity
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As expected, hemoglobin and hematocrit values increased in subjects with TDS after
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administration of TU by an average 2.46 g/dl (p <0.001) and 3.03% (p <0.001), respectively (fig.
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3), representing 18.01% and 7.57% increase, respectively, from baseline. Hb and Hct levels
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peaked at week 18 (visit 4). Thereafter, levels decreased linearly from week 18 to a value of
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15.05 ± 1.54 g/dl and 43.04 ± 3.71 % after the sixth injection (visit 7), however still higher than
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those of values at baseline. The prevalence of anemia decreased in total subjects with TDS after
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administration of TU from 29.6% (baseline) to 10.0% (after visit 7) (p <0.001) (fig. 3). The
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prevalence of anemia decreased from 39.4% (baseline) to 7.7% (visit 7) (p <0.001) in the group
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≥65 years of age from 21.1% (baseline) to 14.3% (visit 7) (p <0.001) in the group <65 years of
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age.
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TU treatment was associated with higher WBV levels (fig. 4). A maximal increase was
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observed at week 18 (visit 4) at shear rate 5 s-1, 50 s-1, 150 s-1 and 300 s-1. Thereafter, the findings
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are consistent with that of the Hct (rs = 0.85). However, WBV and Hct stabilized after 18 weeks
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(visit 5).
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Effects of Testosterone Administration on Anemic Factors
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As demonstrated in figure 5, patients with anemia showed a significant increase in erythropoietin
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levels (p = 0.047) from the baseline to the week 6 (visit 3) and levels of Fe (p = 0.043), ferritin (p
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= 0.031) were significantly decreased from the baseline to the week 18 (visit 4), however slowly
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increased after week 18. Unsaturated iron binding capacity (p = 0.026) and total iron binding
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capacity (p = 0.033) were significantly increased from the baseline to the week 18 (visit 4) but
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slowly increased after week 18. Vitamin B12 levels decreased and folate values increased,
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linearly from the baseline to the end of study (fig. 5).
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Effects of Testosterone Therapy on Lipid Profiles
TC level significantly decreased from 165.89 ± 39.16 mg/dl to 153.80 ± 154.27 mg/dl (p = 0.002)
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after TU therapy. Changes in LDL levels did not reach statistical significance, however, as a
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slight decrease from the baseline (-2.59 ± 8.88) was noted (fig. 6).
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Effects of Testosterone Administration on Anthropometry
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As demonstrated in figure 7, patients showed a significant increase in body weight (p <0.001),
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body mass index (p <0.001), amount of muscle (p <0.001) from the baseline at the week 54 (visit
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7), most likely due to the anabolic effects of testosterone. Patients also reported a decreased from
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the baseline in amount of fat (p = 0.003) and ratio of fat (p <0.001).
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Effects of Testosterone Therapy on Inflammation Profiles
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As demonstrated in figure 8, hs-CRP (p <0.001) and ESR (p <0.001) levels significant decreased
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from the baseline to the week 54 (visit 7).
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DISCUSSION
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The prevalence of low testosterone levels rises from 20% to 50% from the sixth to ninth decades
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of life, respectively.14,15 Epidemiological researches demonstrated that both low and overly high
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testosterone levels were associated with higher risk of CVD.16-18 The recent large observational
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cohort study demonstrated that normalization of TT levels after TRT was associated with a
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significant reduction in all-cause mortality, myocardial infarction, and stroke.19 However, the
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role of TRT on risks of cardiovascular health and metabolic syndrome remains a point of
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concern.
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Although participants with low testosterone levels were not always anemic, the association
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between low testosterone level and lowered hemoglobin level was statistically significant.5 These
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results suggested that a low testosterone level is the one of the causal factor for anemia. Because
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testosterone play an important role in erythropoiesis by stimulates the production of
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erythropoietin-responsive cells and burst forming unit in bone marrow.7 It may be that a low
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testosterone level may be a cause for anemia and our results suggest that TRT in hypogonadal
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patients may be effective in reducing the anemia and CVD associated with anemia.
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In our study, patients with anemia showed a significant increase in erythropoietin levels
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from baseline to week 6. These results suggest that TRT effects on anemia may be via increasing
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erythropoietin levels. Since testosterone stimulates erythropoiesis, Fe supply to the erythron
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requires efficient recycling of Fe. The sources of Fe are macrophages following destruction of
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senescent red blood cells and absorption of Fe from the diet by duodenal enterocytes.20 However
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from our results, with TRT therapy in TDS patients with anemia, Fe and ferritin levels still
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decreased from baseline to week 18 and thus TRT may not be helping Fe recycling as would be
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required for fully supporting recovery from anemia. What inflammatory markers hs-CRP and
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ESR significantly decreased from baseline to week 54 after TRT suggests TRT may also be
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indirect via reducing baseline inflammation.21
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In our study, the 54-week testosterone therapy reduced total cholesterol. This effect may
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have clinical importance as a reduction in TC may lead to a reduced CVD risk. LDL levels did
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not reach statistical significance, nevertheless a slight decrease from the baseline. The reduction
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of TC and LDL levels revealed that TRT improved overall lipid profiles in subjects with TDS
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and potentially helped lower the risk of CVD. Our data confirmed previous observations of lipid
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profile improvement in response to TRT.22 Few studies have investigated the effects TRT on Vit
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D. TRT increased circulating Vit D and its availability.23,24
In our study, patients showed a significant increase in body weight and BMI from the
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baseline at the end of the study. However long term TRT studies have consistently shown the
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weight loss and reduced of BMI.15 Follow-up period of 54 weeks is relatively short for
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assessment of the effect of TRT to body weight and BMI. Also, the subjects in the present study
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were not obese, and it may influence on results.
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Our studies found that short-term testosterone increased hematocrit and whole blood
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viscosity. As hematocrit and whole blood viscosity remained low after week 18 and stabilized at
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54 weeks suggests the presence of adaptive mechanism from the initial changes. The role of
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changes in blood rheology with respect to androgen replacement therapy has not been fully
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investigated and any direct impacts of increased hematocrit and whole-blood viscosity from TRT
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on risks of CVD needs to be fully explored. However, TRT was well tolerated by patients and
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showed no CVD. Blood circulation may be balanced by the vasodilatory effects and erythrocyte
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deformability of testosterone.10
As far as the limitations of our study, it was not a randomized controlled study. This study
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was performed at a single-center and it was an open-label, registry of patients. Also unintended
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bias could have been introduced as the subjects were seeking treatment for various urological
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conditions. In addition, the follow-up period of 54 weeks is relatively short for assessment of our
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findings. Therefore, a randomized, long-term study of TRT will be required. In our study, the
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subjects had to have a total testosterone level of <2.35 ng/ml. We planned to conduct our study at
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before 2011 and in that time, more advanced guidelines such as the recent International Society
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for the Study of the Aging Male guidelines were not published.25,26 It is the limitation of our
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study. Therefore, further study with the recent guidelines will be required to support the findings
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from our study. Even with the above limitations, we believe that our findings show a direct
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benefit of TRT for TDS patients on several parameters tested for the duration of the study. Also,
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our results show that benefits of TRT are not different regardless of age.
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CONCLUSION
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In our study a 54-week TRT for TDS males decreased the prevalence of anemia improving lipid
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profiles and lowering the risk for CVD. Because WBV, Hb and Hct were increased from
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baseline to 18 weeks after TRT but decreased and remained unchanged for the remainder of the
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TU therapy, TRT may not lead to a higher risk of CVD. However since rheological changes may
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prognosticate future vascular events, we recommend further study with longer-term follow-up.
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ACKNOWLEDGEMENTS
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This study was supported by grants from the Biomedical Research Institute of Chonbuk National
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University Hospital, Jeonju, Korea. However, Biomedical Research Institute of Chonbuk
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National University Hospital, Jeonju, Korea had no role in design or conduct of the study. This
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included collection, management, analysis, or interpretation of the data in addition to preparation,
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review, or approval of the manuscript. Professor Jae Hyun Lee supported this study with the
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evaluation of the laboratory medicine.
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undecanoate on metabolic and hormonal parameters in ageing men with metabolic syndrome.
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Int J Endocrinol 2014; 2014: 527470.
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25. Lunenfeld B, Mskhalaya G, Zitzmann M et al: Recommendations on the diagnosis, treatment
and monitoring of hypogonadism in men. Aging Male 2015; 18: 5.
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26. Bhasin S, Pencina M, Jasuja GK et al: Reference ranges for testosterone in men generated
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using liquid chromatography tandem mass spectrometry in a community-based sample of
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healthy nonobese young men in the Framingham Heart Study and applied to three
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geographically distinct cohorts. J Clin Endocrinol Metab 2011; 96: 2430.
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FIGURE LEGENDS
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Figure 1. Schedule of testosterone undecanoate (TU, Nebido®).
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Figure 2. Levels of total testosterone, free testosterone and estradiol with testosterone
4
undecanoate (TU, Nebido®). Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 3. Hemoglobin and hematocrit changes with testosterone undecanoate (TU, Nebido®)
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therapy. Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 4. Whole blood viscosity at shear rates 5 s-1, 50 s-1, 150 s-1, and 300 s-1 following
8
testosterone undecanoate (TU, Nebido®) therapy. cP, centipoises. Single asterisk indicates p
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<0.05 vs. baseline (visit 2). Double asterisks indicate p <0.01 vs. baseline (visit 2).
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Figure 5. Anemic factor (Eythropoietin, UIBC, TIBC, Fe, Ferritin, VitB12, Folate) with
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testosterone undecanoate (Nebido®) therapy.TIBC, total iron binding capacity. VitB12, vitamin
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B12. Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 6. Lipids profile with testosterone undecanoate (TU, Nebido®) therapy. Cholesterol, total
14
cholesterol. HDL, high density lipoprotein. LDL, low density lipoprotein. TG, triglyceride.
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Figure 7. Effects of testosterone undecanoate (TU, Nebido®) therapy on 54-week change in
16
anthropometry. BMI, body mass index. Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 8. hs-CRP and ESR with testosterone undecanoate (Nebido®) therapy. Single asterisk
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indicates p <0.05 vs. baseline (visit 2).
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Table 1. Demographics and baseline characteristic for all enrolled patients Group
Age ≤65 years (19) 47.21 ± 11.04 (17-60)
(No.) Age, years (range)
Age >65 years (10) 68.03 ± 5.65 (61-84)
Total (29) 57 ± 13.65, (17-84)
p value
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Anthropometry Weight, kg 74.27 ± 12.49 67.12 ± 10.36 71.05 ± 12.04 0.021 BMI, kg/m2 26.18 ± 2.78 24.57 ± 3.00 25.46 ± 2.97 0.036 Amount of muscle, kg 49.48 ± 7.21 46.65 ± 5.01 48.21 ± 6.42 0.091 Amount of fat, kg 21.89 ± 7.06 17.40 ± 5.92 19.87 ± 6.89 0.011 Ratio of abdominal fat, % 0.91 ± 0.04 0.88 ± 0.04 0.89 ± 0.04 0.044 Ratio of fat, % 28.94 ± 6.49 25.45 ± 5.89 27.37 ± 6.42 0.035 Hormones Total testosterone, ng/ml 1.51 ± 0.95 2.24 ± 1.13 1.87 ± 1.09 0.005 Free testosterone, pg/ml 2.72 ± 1.74 3.40 ± 2.28 3.04 ± 2.03 0.174 Estradiol, pg/ml 17.06 ± 13.18 18.46 ± 10.75 17.69 ± 12.1 0.197 Lipids profiles Total cholesterol, mg/dl 183.91 ± 38.77 145.66 ± 28.59 166.88 ± 37.56 <0.001 HDL, mg/dl 46.71 ± 10.37 40.82 ± 10.20 43.92 ± 10.62 0.032 LDL, mg/dl 108.45 ± 39.17 76.14 ± 22.91 93.19 ± 36.09 <0.001 Triglycerides, mg/dl 114.77 ± 39.71 124.09 ± 53.53 119.22 ± 46.69 0.419 Erythropoiesis/blood count Hematocrit, % 41.32 ± 4.51 38.54 ± 4.32 40.01 ± 4.61 0.011 Hemoglobin, g/dl ≤ 13 11.65 ± 1.09 11.44 ± 1.19 11.52 ± 1.13 0.077 > 13 14.75 ± 0.94 14.09 ± 0.77 14.47 ± 0.92 Whole blood viscosity, cP Shear rate 5/s-1 10.94 ± 1.96 10.59 ± 1.83 10.78 ± 1.89 0.439 -1 Shear rate 50/s 5.31 ± 0.65 5.25 ± 0.77 5.28 ± 0.71 0.710 Shear rate 150/s-1 4.38 ± 0.68 4.38 ± 0.51 4.38 ± 0.61 0.991 Shear rate 300/s-1 4.08 ± 0.48 4.01 ± 0.45 4.05 ± 0.46 0.533 All the values are expressed as mean ± standard deviation. No., number of patients. BMI, body mass index. HDL, high density lipoprotein. LDL, low density lipoprotein. TG, triglyceride Hemoglobin is stratified into two subgroup (hemoglobin ≤ 12.9 or > 13). cP, centipoises.
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Randomization (N = 58)
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18 weeks
30 weeks
42 weeks
54 weeks
55 weeks
Visit 4
Visit 5
Visit 6
Visit 7
Visit 8 Confirm the laboratory data
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0 week
6 weeks
Visit 1
Visit 2
Visit 3
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Treatment period; Testosterone undecanoate 1 g (N = 29)
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CVD = cardiovascular diseases ESR = erythrocyte sedimentation rate
Hb = hemoglobin Hct = hematocrit LDL = low density lipoprotein hs-CRP = high sensitivity C-reactive protein
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FT = free testosterone
TDS = testosterone deficiency syndrome TRT = testosterone replacement therapy TT = total testosterone
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WBV = whole blood viscosity
PII: DOI: Reference:
S0022-5347(15)05150-2 10.1016/j.juro.2015.10.130 JURO 13046
To appear in: The Journal of Urology Accepted Date: 15 October 2015 Please cite this article as: Zhang LT, Shin YS, Kim JY, Park JK, Could Testosterone Replacement Therapy in Hypogonadal Men Ameliorate Anemia, a Cardiovascular Risk Factor? An Observational, 54week Cumulative Registry Study, The Journal of Urology® (2015), doi: 10.1016/j.juro.2015.10.130. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
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Could Testosterone Replacement Therapy in Hypogonadal Men Ameliorate Anemia, a
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Cardiovascular Risk Factor? An Observational, 54-week Cumulative Registry Study
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Li Tao Zhang,* Yu Seob Shin,* Ji Yong Kim, Jong Kwan Park†
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Department of Urology, Chonbuk National University and Research Institute of Clinical
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Medicine of Chonbuk National University-Biomedical Research Institute and Clinical Trial
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Center of Medical Device of Chonbuk National University, Jeonju, 561-712, Republic of Korea.
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*
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Enclosed: 8 figures and 1 table
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Word count of text/abstract: 2499/250
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Equal study contribution
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Running Title: Effect of testosterone undecanoate therapy on CVD risk factors.
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†
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Jong Kwan Park, M.D., Ph.D.,
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Department of Urology, Chonbuk National University and Research Institute of Clinical
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Medicine of Chonbuk National University-Biomedical Research Institute and Clinical Trial
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Center of Medical Device of Chonbuk National University, Jeonju, 561-712, Republic of Korea.
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Tel.: + 82 (0) 63 250 1510
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Fax: + 82 (0) 63 250 1564
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Correspondence to:
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E-mail: [email protected]
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Purpose: This study was to investigate if testosterone undecanoate in patients with
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hypogonadism attenuates anemia and the risk for cardiovascular disease.
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Materials and Methods: A registry study consisted of 58 participants with subnormal total
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testosterone level (<2.35 ng/ml) and at least mild symptoms of testosterone deficiency. All the
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patients were injected with 1,000 mg of testosterone undecanoate on initial visit, followed by
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injection at 6, 18, 30, 42 and 54 weeks. Serum hormones, Hb, Hct, anemia risk factors, lipid
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profiles, whole blood viscosity and anthropometry were measured.
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Results: Total testosterone (from 1.87 ± 1.09 to 5.52 ± 1.92 ng/ml, p <0.001) and free
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testosterone (from 3.04 ± 2.03 to 7.23 ± 2.90 pg/ml, p <0.001) were restored by testosterone
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undecanoate therapy. Hb and Hct significantly increased after testosterone undecanoate therapy
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by an average of 2.46 g/dl (p <0.001) and 3.03% (p <0.001), respectively. Prevalence of anemia
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(from 29.6 to 10.0%) significantly decreased (p <0.001) and patients with anemia showed a
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significant increase in erythropoietin (p = 0.047) after testosterone undecanoate therapy.
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Reduction in total cholesterol (from 165.89 ± 39.16 to 153.80 ± 154.27 mg/dl, p = 0.002), higher
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whole blood viscosity and increased Hct were observed until 54 weeks compared with baseline,
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however whole blood viscosity and Hct stabilized after 18 weeks.
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Conclusions: 54-week testosterone undecanoate decreased the prevalence of anemia and
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components of metabolic syndrome. A longer duration testosterone undecanoate therapy of more
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than 18 weeks may be effective and safe in reducing the blood viscosity and improving anemia.
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Key Words: anemia; cardiovascular diseases; hypogonadism; whole blood visccosity; testosterone
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MALE hypogonadism results from failure of testes to produce adequate sex hormones; this may
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be because of distant or local hormonal deficiency.1 Normal aging could be one of the causes of
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the deficiency. Collectively referred to as TDS, the associated symptoms may be erectile
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dysfunction, increased abdominal fat and reduced muscle.2 Low circulating testosterone is also
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linked to conditions such as anemia, metabolic syndrome, and CVD.3
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From the Massachusetts Male Aging Study of older men (years 1987-2004), the average
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age-related reduction in TT is 0.8~1.6% per year after age 40.4 For hypogonadal men, anemia is
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a frequent finding as testosterone stimulates erythropoiesis in a dose dependent manner and more
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prominently in older men.5 This is despite the effect being independent of actual erythropoietin
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and transferrin receptor levels.5-7 It is reasonable to hypothesize that a decline in testosterone
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levels in males with TDS may lead to an anemia and increase the risk for CVD.
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The mechanism linking TDS with metabolic sydrome seems to be complicated and multi-
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directional.8,9 It is known that metabolic sydrome is associated with an increasing risk of CVD.
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Hematological parameters such as increased whole blood viscosity (WBV), increased
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erythrocyte aggregation, reduced erythrocyte deformability, and altered erythrocyte morphology
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have also been associated with metabolic syndrome and CVD.10-12
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The aims of the current study were 1) to investigate whether a TU formulation, Nebido®,
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has a sustained benefit on anemia, metabolic and CVD parameters in men with TDS; and 2) to
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measure any dynamic alterations in blood viscosity by TU and its relationship to the risk of CVD
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in patients with TDS. Nebido® was approved as AVEED (3 ml of TU) by the FDA in the United
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State.
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MATERIALS AND METHODS
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Ethics and Informed Consent
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This study was conducted at Chonbuk National University Hospital. Written informed consent
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was obtained from all subjects before study enrollment. Study protocols and informed consent
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forms were approved by the Institutional Review Board (IRB; Number, CUH 2015-06-133-001)
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(Clinical Research Information Service; CRIS, KCT0001680).
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Study Design
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This study was an open-labeled, cumulative analysis of adult males with TDS. The study was
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conducted from February 2012. Last subject completed the study at June 2015. Parameters were
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measured during the initial screening visit (visit 1), followed by measurements at 6, 18, 30, 42,
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and 54 weeks after the initial injection (fig. 1).
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Subjects
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The study population was comprised of hypogonadal patients who were elected to participate in
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the open-label, cumulative registry study. The subjects were male aged 18 to 80 years and had a
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body mass index of ≥ 18 to ≤ 40 kg/m2. The individuals had to have a serum TT level < 2.35
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ng/ml and at least mild TDS to be included in the study. Most hypogonadal men had not received
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any previous androgen treatment, and those who had, had to have had discontinued testosterone
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treatment for at least 6 months prior to enrollment in the study. The patients had a rectal
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examination, transrectal ultrasonography and their prostate-specific antigen levels were checked
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before treatment.
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The enrolled group (n = 58) received active treatment during the open phase of the study. 29
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patients of them were dropped out before visit 3 due to low economic state (22), or without
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improvement of symptoms (7). The patients were injected with 1,000 mg of TU on ventro-
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gluteal muscle of hip on initial visit, followed by injection at 6, 18, 30, 42 and 54 weeks.
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The exclusions were for: tobacco smokers, prostate-specific antigen level of >2.5 ng/ml, a
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current or previous diagnosis of prostate cancer, International Prostate Symptom Score of >15,
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an untreated prolactinoma, Hct of >52% or Hb >18 g/dl, having a uncontrolled diabetes mellitus
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(HbA1c above 10%), having liver and kidney disease, having known diseases of the testis or the
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pituitary.
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General and Biochemical Analyses
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A venous blood sample was drawn for complete blood count, TC, triglyceride, high density
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lipoprotein, LDL, fibrinogen, glucose, hs-CRP, and erythrocyte sedimentation rate (ESR).
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Measurement of Hormones
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TT evaluations were always made between 8 and 10 am. Hormonal tests included TT (COAT-A-
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COUNT, Siemens Healthcare Diagnostics Inc, Los Angeles, CA, USA), FT (Free Testosterone,
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COAT-A-COUNT, Siemens Healthcare Diagnostics Inc,), and estradiol (Estradiol, BECKMAN
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COULTER, Prague, Czech). TT level <2.35 (2.36< TT< 9.96 ng/ml, FT <8.7 (8.8< FT <27)
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pg/ml were considered to be low. The reference range of estradiol is >7.6 and <42.6 pg/ml.
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Measurement of Whole Blood Viscosity
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Viscosity measurement was made at shear rates of 1, 2, 5, 10, 50, 150, 300, and 1,000 sec-1
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samples.13 These shear rates include the velocity of blood flows in the circulatory system.
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Anemic Outcome Measures
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Blood sample levels of iron, ferritin, unsaturated iron binding capacity, total iron binding
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capacity, vitamin B12, folate and erythropoietin were determined.
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Other Main Outcome Measures
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Anthropometry parameters were determined by body composition analyzer.
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Statistical Analysis 7
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Only patients who had completed 54 weeks of treatment were eligible for evaluation.
Statistical analysis was performed using the Statistical Package for Social Sciences for
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Windows, version 21 (SPSS, Chicago IL, USA). All values were checked for normal distribution
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by the Kolmogorov-Smirnov for goodness of fit. Descriptive variables are presented as mean ±
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standard deviation. One way analysis of variance was calculated over time, which was followed
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by the Duncan’s post-hoc test for intergroup comparison if an overall level of significance of p
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<0.05 was reached.
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RESULTS
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Demographics and Comorbidities of the Participants at Baseline
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The 29 subjects completed the 54-week intervention and were assessed at baseline (visit 2), after
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6 weeks (visit 3), 18 (visit 4), 30 (visit 5), 42 (visit 6), 54 (visit 7), and 1 week after
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discontinuation of intervention (visit 8). A number of comorbidities in the patients were reported
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in this registry study. From among the 58 randomized patients there were 27 cases (46.6%) with
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comorbidities. 29.31% of these had type 2 diabetes mellitus, 37.93% had hypertension, 19.0%
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had hypertension and type 2 diabetes together and 22.4% had coronary artery diseases. A small
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percentage (<10%) suffered from elevated triglycerides (4.9%) and dyslipidemia (5.2%). During
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the study, there was no patient who need to receive prostate biopsy for work up of prostate
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cancer. Also, there were no major side effects occurred during the study. Baseline demographic
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and clinical characteristics are shown in table 1.
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Serum Total, Free Testosterone, and Estradiol Profile over 54 Weeks after Treatment
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Serum TT, FT, and estradiol values over the 54 weeks (after visit 7) are shown in figure 2. Pre-
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dose concentrations were 1.87 ± 1.09 ng/ml, 3.04 ± 2.03 pg/ml and 25.22 ± 2.84 pg/ml,
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respectively. After first application of testosterone, TT and FT reads rose rapidly, reaching 5.99
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± 1.97 ng/ml (p <0.001) and 9.13 ± 3.41 pg/ml (p <0.001), respectively. Levels decreased
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linearly from week 6 to a value of 5.45 ± 2.47 ng/ml before the third injection. Thereafter, serum
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TT and FT concentrations slightly altered, but still remained the eugonadal range (2.36-9.96
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ng/ml and 8.8-27 pg/ml) until 54 weeks post-application. Estradiol slowly and significantly
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increased, reaching 25.22 ± 14.77 pg/ml (p <0.001) at visit 4.
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Effects of Testosterone Therapy on Hemoglobin, Hematocrit, and Whole Blood Viscosity
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As expected, hemoglobin and hematocrit values increased in subjects with TDS after
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administration of TU by an average 2.46 g/dl (p <0.001) and 3.03% (p <0.001), respectively (fig.
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3), representing 18.01% and 7.57% increase, respectively, from baseline. Hb and Hct levels
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peaked at week 18 (visit 4). Thereafter, levels decreased linearly from week 18 to a value of
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15.05 ± 1.54 g/dl and 43.04 ± 3.71 % after the sixth injection (visit 7), however still higher than
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those of values at baseline. The prevalence of anemia decreased in total subjects with TDS after
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administration of TU from 29.6% (baseline) to 10.0% (after visit 7) (p <0.001) (fig. 3). The
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prevalence of anemia decreased from 39.4% (baseline) to 7.7% (visit 7) (p <0.001) in the group
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≥65 years of age from 21.1% (baseline) to 14.3% (visit 7) (p <0.001) in the group <65 years of
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age.
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TU treatment was associated with higher WBV levels (fig. 4). A maximal increase was
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observed at week 18 (visit 4) at shear rate 5 s-1, 50 s-1, 150 s-1 and 300 s-1. Thereafter, the findings
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are consistent with that of the Hct (rs = 0.85). However, WBV and Hct stabilized after 18 weeks
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(visit 5).
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Effects of Testosterone Administration on Anemic Factors
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As demonstrated in figure 5, patients with anemia showed a significant increase in erythropoietin
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levels (p = 0.047) from the baseline to the week 6 (visit 3) and levels of Fe (p = 0.043), ferritin (p
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= 0.031) were significantly decreased from the baseline to the week 18 (visit 4), however slowly
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increased after week 18. Unsaturated iron binding capacity (p = 0.026) and total iron binding
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capacity (p = 0.033) were significantly increased from the baseline to the week 18 (visit 4) but
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slowly increased after week 18. Vitamin B12 levels decreased and folate values increased,
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linearly from the baseline to the end of study (fig. 5).
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Effects of Testosterone Therapy on Lipid Profiles
TC level significantly decreased from 165.89 ± 39.16 mg/dl to 153.80 ± 154.27 mg/dl (p = 0.002)
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after TU therapy. Changes in LDL levels did not reach statistical significance, however, as a
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slight decrease from the baseline (-2.59 ± 8.88) was noted (fig. 6).
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Effects of Testosterone Administration on Anthropometry
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As demonstrated in figure 7, patients showed a significant increase in body weight (p <0.001),
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body mass index (p <0.001), amount of muscle (p <0.001) from the baseline at the week 54 (visit
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7), most likely due to the anabolic effects of testosterone. Patients also reported a decreased from
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the baseline in amount of fat (p = 0.003) and ratio of fat (p <0.001).
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Effects of Testosterone Therapy on Inflammation Profiles
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As demonstrated in figure 8, hs-CRP (p <0.001) and ESR (p <0.001) levels significant decreased
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from the baseline to the week 54 (visit 7).
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DISCUSSION
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The prevalence of low testosterone levels rises from 20% to 50% from the sixth to ninth decades
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of life, respectively.14,15 Epidemiological researches demonstrated that both low and overly high
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testosterone levels were associated with higher risk of CVD.16-18 The recent large observational
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cohort study demonstrated that normalization of TT levels after TRT was associated with a
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significant reduction in all-cause mortality, myocardial infarction, and stroke.19 However, the
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role of TRT on risks of cardiovascular health and metabolic syndrome remains a point of
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concern.
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Although participants with low testosterone levels were not always anemic, the association
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between low testosterone level and lowered hemoglobin level was statistically significant.5 These
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results suggested that a low testosterone level is the one of the causal factor for anemia. Because
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testosterone play an important role in erythropoiesis by stimulates the production of
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erythropoietin-responsive cells and burst forming unit in bone marrow.7 It may be that a low
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testosterone level may be a cause for anemia and our results suggest that TRT in hypogonadal
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patients may be effective in reducing the anemia and CVD associated with anemia.
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In our study, patients with anemia showed a significant increase in erythropoietin levels
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from baseline to week 6. These results suggest that TRT effects on anemia may be via increasing
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erythropoietin levels. Since testosterone stimulates erythropoiesis, Fe supply to the erythron
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requires efficient recycling of Fe. The sources of Fe are macrophages following destruction of
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senescent red blood cells and absorption of Fe from the diet by duodenal enterocytes.20 However
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from our results, with TRT therapy in TDS patients with anemia, Fe and ferritin levels still
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decreased from baseline to week 18 and thus TRT may not be helping Fe recycling as would be
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required for fully supporting recovery from anemia. What inflammatory markers hs-CRP and
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ESR significantly decreased from baseline to week 54 after TRT suggests TRT may also be
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indirect via reducing baseline inflammation.21
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In our study, the 54-week testosterone therapy reduced total cholesterol. This effect may
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have clinical importance as a reduction in TC may lead to a reduced CVD risk. LDL levels did
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not reach statistical significance, nevertheless a slight decrease from the baseline. The reduction
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of TC and LDL levels revealed that TRT improved overall lipid profiles in subjects with TDS
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and potentially helped lower the risk of CVD. Our data confirmed previous observations of lipid
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profile improvement in response to TRT.22 Few studies have investigated the effects TRT on Vit
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D. TRT increased circulating Vit D and its availability.23,24
In our study, patients showed a significant increase in body weight and BMI from the
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baseline at the end of the study. However long term TRT studies have consistently shown the
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weight loss and reduced of BMI.15 Follow-up period of 54 weeks is relatively short for
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assessment of the effect of TRT to body weight and BMI. Also, the subjects in the present study
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were not obese, and it may influence on results.
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Our studies found that short-term testosterone increased hematocrit and whole blood
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viscosity. As hematocrit and whole blood viscosity remained low after week 18 and stabilized at
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54 weeks suggests the presence of adaptive mechanism from the initial changes. The role of
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changes in blood rheology with respect to androgen replacement therapy has not been fully
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investigated and any direct impacts of increased hematocrit and whole-blood viscosity from TRT
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on risks of CVD needs to be fully explored. However, TRT was well tolerated by patients and
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showed no CVD. Blood circulation may be balanced by the vasodilatory effects and erythrocyte
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deformability of testosterone.10
As far as the limitations of our study, it was not a randomized controlled study. This study
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was performed at a single-center and it was an open-label, registry of patients. Also unintended
5
bias could have been introduced as the subjects were seeking treatment for various urological
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conditions. In addition, the follow-up period of 54 weeks is relatively short for assessment of our
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findings. Therefore, a randomized, long-term study of TRT will be required. In our study, the
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subjects had to have a total testosterone level of <2.35 ng/ml. We planned to conduct our study at
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before 2011 and in that time, more advanced guidelines such as the recent International Society
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for the Study of the Aging Male guidelines were not published.25,26 It is the limitation of our
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study. Therefore, further study with the recent guidelines will be required to support the findings
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from our study. Even with the above limitations, we believe that our findings show a direct
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benefit of TRT for TDS patients on several parameters tested for the duration of the study. Also,
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our results show that benefits of TRT are not different regardless of age.
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CONCLUSION
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In our study a 54-week TRT for TDS males decreased the prevalence of anemia improving lipid
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profiles and lowering the risk for CVD. Because WBV, Hb and Hct were increased from
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baseline to 18 weeks after TRT but decreased and remained unchanged for the remainder of the
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TU therapy, TRT may not lead to a higher risk of CVD. However since rheological changes may
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prognosticate future vascular events, we recommend further study with longer-term follow-up.
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ACKNOWLEDGEMENTS
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This study was supported by grants from the Biomedical Research Institute of Chonbuk National
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University Hospital, Jeonju, Korea. However, Biomedical Research Institute of Chonbuk
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National University Hospital, Jeonju, Korea had no role in design or conduct of the study. This
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included collection, management, analysis, or interpretation of the data in addition to preparation,
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review, or approval of the manuscript. Professor Jae Hyun Lee supported this study with the
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evaluation of the laboratory medicine.
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1. Corona G, Rastrelli G, Forti G et al: Update in testosterone therapy for men. J Sex Med 2011; 8: 639.
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FIGURE LEGENDS
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Figure 1. Schedule of testosterone undecanoate (TU, Nebido®).
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Figure 2. Levels of total testosterone, free testosterone and estradiol with testosterone
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undecanoate (TU, Nebido®). Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 3. Hemoglobin and hematocrit changes with testosterone undecanoate (TU, Nebido®)
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therapy. Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 4. Whole blood viscosity at shear rates 5 s-1, 50 s-1, 150 s-1, and 300 s-1 following
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testosterone undecanoate (TU, Nebido®) therapy. cP, centipoises. Single asterisk indicates p
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<0.05 vs. baseline (visit 2). Double asterisks indicate p <0.01 vs. baseline (visit 2).
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Figure 5. Anemic factor (Eythropoietin, UIBC, TIBC, Fe, Ferritin, VitB12, Folate) with
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testosterone undecanoate (Nebido®) therapy.TIBC, total iron binding capacity. VitB12, vitamin
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B12. Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 6. Lipids profile with testosterone undecanoate (TU, Nebido®) therapy. Cholesterol, total
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cholesterol. HDL, high density lipoprotein. LDL, low density lipoprotein. TG, triglyceride.
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Figure 7. Effects of testosterone undecanoate (TU, Nebido®) therapy on 54-week change in
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anthropometry. BMI, body mass index. Single asterisk indicates p <0.05 vs. baseline (visit 2).
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Figure 8. hs-CRP and ESR with testosterone undecanoate (Nebido®) therapy. Single asterisk
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indicates p <0.05 vs. baseline (visit 2).
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Table 1. Demographics and baseline characteristic for all enrolled patients Group
Age ≤65 years (19) 47.21 ± 11.04 (17-60)
(No.) Age, years (range)
Age >65 years (10) 68.03 ± 5.65 (61-84)
Total (29) 57 ± 13.65, (17-84)
p value
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Anthropometry Weight, kg 74.27 ± 12.49 67.12 ± 10.36 71.05 ± 12.04 0.021 BMI, kg/m2 26.18 ± 2.78 24.57 ± 3.00 25.46 ± 2.97 0.036 Amount of muscle, kg 49.48 ± 7.21 46.65 ± 5.01 48.21 ± 6.42 0.091 Amount of fat, kg 21.89 ± 7.06 17.40 ± 5.92 19.87 ± 6.89 0.011 Ratio of abdominal fat, % 0.91 ± 0.04 0.88 ± 0.04 0.89 ± 0.04 0.044 Ratio of fat, % 28.94 ± 6.49 25.45 ± 5.89 27.37 ± 6.42 0.035 Hormones Total testosterone, ng/ml 1.51 ± 0.95 2.24 ± 1.13 1.87 ± 1.09 0.005 Free testosterone, pg/ml 2.72 ± 1.74 3.40 ± 2.28 3.04 ± 2.03 0.174 Estradiol, pg/ml 17.06 ± 13.18 18.46 ± 10.75 17.69 ± 12.1 0.197 Lipids profiles Total cholesterol, mg/dl 183.91 ± 38.77 145.66 ± 28.59 166.88 ± 37.56 <0.001 HDL, mg/dl 46.71 ± 10.37 40.82 ± 10.20 43.92 ± 10.62 0.032 LDL, mg/dl 108.45 ± 39.17 76.14 ± 22.91 93.19 ± 36.09 <0.001 Triglycerides, mg/dl 114.77 ± 39.71 124.09 ± 53.53 119.22 ± 46.69 0.419 Erythropoiesis/blood count Hematocrit, % 41.32 ± 4.51 38.54 ± 4.32 40.01 ± 4.61 0.011 Hemoglobin, g/dl ≤ 13 11.65 ± 1.09 11.44 ± 1.19 11.52 ± 1.13 0.077 > 13 14.75 ± 0.94 14.09 ± 0.77 14.47 ± 0.92 Whole blood viscosity, cP Shear rate 5/s-1 10.94 ± 1.96 10.59 ± 1.83 10.78 ± 1.89 0.439 -1 Shear rate 50/s 5.31 ± 0.65 5.25 ± 0.77 5.28 ± 0.71 0.710 Shear rate 150/s-1 4.38 ± 0.68 4.38 ± 0.51 4.38 ± 0.61 0.991 Shear rate 300/s-1 4.08 ± 0.48 4.01 ± 0.45 4.05 ± 0.46 0.533 All the values are expressed as mean ± standard deviation. No., number of patients. BMI, body mass index. HDL, high density lipoprotein. LDL, low density lipoprotein. TG, triglyceride Hemoglobin is stratified into two subgroup (hemoglobin ≤ 12.9 or > 13). cP, centipoises.
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Randomization (N = 58)
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18 weeks
30 weeks
42 weeks
54 weeks
55 weeks
Visit 4
Visit 5
Visit 6
Visit 7
Visit 8 Confirm the laboratory data
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Laboratory study
- 4 weeks
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6 weeks
Visit 1
Visit 2
Visit 3
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Treatment period; Testosterone undecanoate 1 g (N = 29)
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CVD = cardiovascular diseases ESR = erythrocyte sedimentation rate
Hb = hemoglobin Hct = hematocrit LDL = low density lipoprotein hs-CRP = high sensitivity C-reactive protein
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TC = total cholesterol
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FT = free testosterone
TDS = testosterone deficiency syndrome TRT = testosterone replacement therapy TT = total testosterone
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TU = testosterone undecanoate (Nebido®)
UIBC = unsaturated iron binding capacity
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WBV = whole blood viscosity